Researchers have adapted a high-magnification technique for viewing body structures at the cellular level so that they can visualize blood vessels in human tumors in real time.

Using the technique, called intravital microscopy (IVM), they found that approximately half of blood vessels in tumors in patients with melanoma had no blood flow and that tumor blood vessels were much larger than would have been anticipated from prior studies.

According to the research team, led by Joseph Skitzki, M.D., of Roswell Park Cancer Institute, the findings have potentially significant implications for the delivery of cancer treatments such as chemotherapy and immunotherapy.

The study was published February 17 in Nature Communications.

Overcoming Technical Challenges

Intravital microscopy has been used in animal models for decades to study drug delivery and cancer biology. But, until recently, the method had not been used in humans due to several technical challenges, including how to illuminate the tumor without interfering with surgery or disrupting the blood supply to the tumor.

Although transillumination, or light transmission through tissue, is generally not possible, Dr. Skitzki and his colleagues were able to address this challenge by using an approach called epifluorescence lighting, which illuminates the tissue from above with a high-intensity light source.

Another key challenge, Dr. Skitzki said, was stabilizing the microscope. Typically, intravital microscopes are set up for use in the laboratory, but the researchers needed to build one that could be moved around the operating room.

“Any vibration is going to cause major changes in the visual field of the microscope, so we had to design something that would be portable but completely stable,” he explained. “With a cantilever design and a heavy base—over 800 lbs.—we created incredible stability at the focus point. Now, with the appropriate software, you can generate very good images at 100 times magnification or higher,” he said.

No Blood Flow

The team designed the microscope and tested it in melanoma patients undergoing surgery to remove a tumor. Their finding that half of the blood vessels in these tumors did not exhibit blood flow at any given time came as a surprise, Dr. Skitzki said. “That would never have been anticipated from pathology slides or anything like that,” he explained.

To make sure this result wasn’t an artifact of the surgical procedure itself (e.g., trauma from the surgery), the researchers examined tumor blood flow in three patients whose skin was so thin that their tumors could be visualized directly through the skin. They found, again, that approximately 50 percent of the blood vessels had no blood flow.

That’s a clinically important finding, explained Dr. Skitzki, because “whether we’re talking about chemotherapy or immunotherapy, half of the inlets to the tumors are closed, so the therapies can’t get to different parts of the tumor.”

Generally, many of the tumor blood vessels also looked different than typical blood vessels, the authors reported. “Tumor vessels were noted by their tortuosity and convoluted architecture, which was in stark contrast to normal linear vessels detected in the surrounding skin,” they wrote.

Further studies will need to answer important questions, such as whether poorly functioning blood vessels in tumors can become functional or what causes them to stop functioning, Dr. Skitzki said. “Because, if you can open up the vasculature, you may make the tumor more accessible to treatment.”

Vessel Size Potentially Affects Treatment

The researchers also found that all of the visualized tumor blood vessels were much larger in diameter than would have been anticipated based on animal studies or from pathology, Dr. Skitzki said.

Blood vessel diameter can potentially affect treatment in several ways, including impeding drug delivery and the efficacy of immunotherapy drugs. For example, the physical forces of the blood flowing through the vessels “are a substantial hurdle to the trafficking of immune cells across the tumor endothelium,” the researchers wrote.

“The study is quite significant and made a number of tantalizing observations that encourage further investigation using such an IVM system,” said Yantian Zhang, Ph.D., of the Imaging Technology Branch in NCI’s Division of Cancer Treatment and Diagnosis. The findings on tumor vessel size, for example, “would require us to re-examine our understanding of tumor blood flow and associated tumor metabolism, drug delivery, and tumor host interaction,” Dr. Zhang said.

The IVM technique, he continued, “could lead to improved understanding of tumor vasculature and tumor microenvironment, and potentially lead to improved patient prognosis and measurement of therapy response.”

Going forward, the team will use the technique to examine a variety of different tumor types, including ovarian tumors. The team also plans to develop ways to better visualize tumor physiology.

“Ultimately, if we can develop fluorescent reagents that can be viewed in real time, perhaps we could visualize drug uptake and delivery to the tumor,” Dr. Skitzki explained. “I think there is definitely the potential to develop reagents that will give you real-time readouts of the tumor, so you can truly personalize the care of the patient.”

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